How Christians Influenced The United States - 1690 Words

A lot has happened over the this course learning everything from music in the 1960s to interrogation techniques of the current. The last ting we learned about our country is how Christians played a role in it. The history of we came to be in America, how we have an influence and the social enigmas with wanting it in politics. The main things we covered in the past eight weeks is how different events have effected or influenced the United States. We begin in the sixties and worked our way through the major events and crises of our country. Sadness could be the label of the sixties, but it wasn t the only thing influencing culture. We often forget one of the most powerful influences of this time and through all of history to now Music is and was powerful. The sixties are a decade that holds many history making changes, from assassination to wars, but looking back you can see what the music was about and it clearly points out why certain things were popular. It was a huge factor in culture and still is today. â€Å"In 1960, nearly half of America s population is under 18 years old† This is what caused music to influence us so greatly the fact that we could get our mind off of war and other horrible things and just let lose. The mood is what has become most important instead of meaning which I think is sad. Looking back you see the Beatles, they sang about girls and how they made them feel. Now in currently you have the boy band One Direction, who is doing the sameShow MoreRelatedReligion : The United States And The European Union1648 Words   |  7 Pagesboth our individual and collective life (Hajizadeh, 2013). Religion plays an integral role in every country whether it is a third-world country, such as some African or Middle Eastern countries, or industrialized capitalist countries like the United States and the European Union. Over the past few decades many global events have brought religion’s growing influence over society, especially its role in affecting politics (Hajizadeh, 2013). The biggest religious event/influence in recent years wouldRead MoreTurning Men Into Fishing Is A Popular Past Time All Around The World1277 Words   |  6 PagesMarch 10, 2015 Turning Men into Fishermen Fishing is a popular past time all around the world. It has been around for many centuries, and has influenced and shaped many societies and cultures. One popular religion that fishing has a major impact on is Christianity. It is obvious that fishing is prominent in the Christian religion by looking at the famous Christian symbol, the Jesus fish. On many American cars one may find this very simple depiction of a fish on the back bumper. Another influence thatRead MoreThe Islamic Faith : Born On The Arabian Peninsula1377 Words   |  6 Pages The Islamic faith was born on the Arabian Peninsula. In the sixth century the Arabian Peninsula was a place of refuge from two major apposing powers the Christian Byzantine kingdom and the Zoroastrain Persian Kingdom (Allen Tooraw, 2011). During this time Mohammad Ibn Abadallah was born. When he was forty years old he had his first vision, proclaiming that Allah was the one true god and rejecting the idol worship of Mecca and thus began the Is lamic faith (Allen Tooraw, 2011). The Quran isRead MoreThe Influence Of The Church1369 Words   |  6 PagesCorinthians 10:31, Paul states the position of the church best: â€Å"So whether you eat or drink, or whatever you do, do it all for the glory of God.† This scripture is an accurate depiction of how the church teaches its followers. Observing the mechanics of how our nation runs, a person can see that religion is the fabric that touches each facet of our country. Three areas where the influence of the church is easily identifiable is government, education, and society. Christian morals and beliefs haveRead MoreChristianity : A Christian Nation1188 Words   |  5 Pagestopics of America being founded as a Christian Nation and the idea of Jesus being followed in America. America was and is Christian, still, but was not founded on Christianity. Fea does not answer the question regarding America being founded as a Christian nation (Fea 245). Prothero gives some opinion about Jesus in America, but says Jesus is viewed different in the eyes of many Americans (Prothero 9). As much as I want to think that America was founded as a Christian nation, I cannot agree with it. TheRead MoreThe Theodosian Code1250 Words   |  5 Pagesway north to what is now known as the United Kingdom. In fact, the empire was so expansive that there was a need for organized law; and so with each emperor there came new constitutions and decrees for the Roman people to follow. The Theodosian Code was just one of the many juristic materials that helped define Roman law and keep legal clarity until the fall of the Byzantine Empire in 1453 C.E.1This paper will define the contents of The Theodosian Code; show how the size and organization of the RomanRead MoreThe Between Mythology And Philosophy1033 Words   |  5 Pagesbearing the weight of all sins, he created new and glorious, beautiful art, by saving every soul. Looking at the arts through both the views of Greek dichotomy and the Biblical view creates an understanding of how the arts might have evolved to what they are today. Art has been vastly influenced by Philosophy. Plato, student of Socrates created his own thoughts about life, art, and reality. In many ways, Plato the Greek dichotomy. Seeing the world through the eyes of someone who believes in one thingRead MoreIslam And The Trans Atlantic Trade1739 Words   |  7 Pagesnot a surprise to find that Islam as a religion was heavily influenced by other religions such as Christianity and Judaism; Muhammad claimed that angel Gabriel visited him with a message from God. The same angel for instance is being referred and Christianity stories. Moses, Abraham and Jesus are also considered as prophets in Islam. The historical advancement of Islam has frequently been connected to the presence of an Islamic state or domain. Hundreds of years after his death, Muhammad s ArabianRead MoreBenefits Of The Spanish Colonization1409 Words   |  6 Pagesreader will learn about what benefits exactly were brought by Spanish Colonization and how some of these benefits, benefit The United States today. So let this journey began. Throughout America’s history there has been many colonizers, explorers, and settlers that came to this part of the world. Spanish in particular began settling in this section of the world, thousands of years ago though it wasn t the United States, but it was one of the Americas. Spain however didn t start thinking like this orRead MoreJohn Winthrop And John Adams1284 Words   |  6 PagesJohn Winthrop and John Adams and their respective works were heavily influenced by their times. Winthrop was a devout Puritan Christian that was escaping a persecuting king in the early to mid-seventeenth century. As a learned and religious man, he came to be the leader of this puritan sect that was to establish a society unlike any other, free from religious corruption that his fellow people saw in the Church of England, that would be a model community much like the â€Å"Citty upon a Hill† described

The Olivet Boys And Girls Club - 1099 Words

The Olivet Boys and Girls Club is a positive place for kids. It allows kids to build leadership skills while interacting with their peers and staff. You can join the Olivet if you are between the ages of 6-18, and with an easy payment of $15 for the academic year. The Olivet is run by caring staff to help you every step of the way if you are needing help. For example if you would need help with your homework, the staff will be there to help. They also have fun activities for you to participate in; like a basketball team, art club, and Keystone/Torch club. Everyone has many stories to tell at the Boys and Girls Club, this is my basketball journey. I have been a member of the Olivet Boys and Girls Club for nine years; starting at nine. Throughout those years, they have taught me the many things that I put into play today. Spending most, if not all of my time at the Olivet’s, I tend to call it my second home. My mother used to always yell at me for not being home all the time. I just loved being there all the time. Through all the support of the Olivet family, I have been able to overcome many obstacles. For example, many may think that because of my height I would have picked basketball as my go to sport; however, that was not the case at all. Luckily, I had a unit director who would not take no for an answer and kept pushing me. Mrs. Deja Harris (Ms. Dede) was my unit Director for the perk unit that I was at. She has become like a second mother figure to me. She was always

The temple architecture of ancient Greece and Rome Free Essays

Brought down from Mount Pinstripes, it was then transported ten miles on oxen-drawn carts to Athens. 5 The Parthenon was regarded in its prime as the finest example of a Doric temple, due mainly to the refinement of already well establish deed aspects of appeal. 6 Typically, only one order was used in the construction of temples in certain areas; Doric in western and mainland Greece, and Ionic on the coast of Asia Minor and in the Aegean islands. We will write a custom essay sample on The temple architecture of ancient Greece and Rome or any similar topic only for you Order Now The Parthenon is an exception to this, however, as it contains certain Ionic elements throughout its structure also, making it unique. The home to an Ionic frieze, the Parthenon had two rooms, most likely in accordance with the sun – one west- facing that acted as a treasury, and a second separate room with its door facing east, used to contain the â€Å"cult image, the gold and ivory Athena Parthenon†, the Greek goddess of reason, intelligent activity, arts and literature. 8 Horizontal aspects such as the architrave and the astrolabe are corrected by meaner of entities, so that they do not â€Å"sag† in the middle. Also, the corner columns stand closer and thicker than their neighbors, so that they do not appear frail against the sky. It is clear that the Greeks at the time were highly skilled in both construction and aesthetic design. 3. Henry Sterile, Greece; From Mycenae to the Parthenon, Teaches Press, First Edition, 2001. P. 189 4. Robin Francis Rhodes, Architecture and Meaning on the Athenian Acropolis, Cambridge University Press, First Edition, 1998. P. 1 5. Bernard Assemble, Architect and Sculptor in Classical Greece; The Wrights Lectures: Volume 6, Phaeton Press, First Edition, 1972. . 94 6. Http://en. Wisped. Org/wick/ Temple_of_Hyphenates 7. A. W. Lawrence, Greek Architecture, Yale University Press, Fifth Edition, 1996. . 77 8. Http://www. Geochronology. Com/Olympian/Athena/Athena. HTML 9. Patrick Mutagens, The Story of Architecture, Phaeton Press, Second Edition, 2004. P. 97 Doric architecture, further contributing to its aesthetic AAA? ¬@lllustration 5: The Parthenon, Acropolis, Athens. Built from 427 – 424 BCC, the Temple of Athena Nikkei is the first fully Ionic structure to be built atop the Athenian Acropolis. As a result, certain differences exist between itself and the previous Parthenon. Measuring a mere 11 feet in heighten, it has been described as â€Å"barely more than a treasury. 11 Of considerable elegance, the temple’s four- columned inappropriately facades acted to frame a small square â€Å"NAS† measuring mm by 5, the entrance to which was preceded by two slender marble pillars. 12 The bastion face looking outwards and the end of the temple tilt towards the north wing, resulting in an altered view of the distance between the wings when viewed from the west. This also forces the corner of the facade in the north-west into line with the south wing facade. This in turn makes the temple sides point towards the Parthenon. 13 Although different techniques were used, it is clear that visual manipulation was Just as prominent in the Temple of Athena Nikkei as it was in the Parthenon. As on red and black pottery, the ancient Greeks favored contrasts, especially those found in nature, and this was readily apparent through their use of marble on the frieze below the temple. 14 The temple itself, similar to the Parthenon and other Doric temples, was built using white phonetic marble. It was, however, built in stages, as war was present during the construction, and from time to time funds were cut short. The sculptural works found on the parapet consist of multiple Nines leading bulls to be sacrificed to Athena ND organizing trophies of victory. It is clear that such ideals were valued by the people of the time, and is the reason why such a great deal of effort was made when constructing temples, and their decorative features. This differs from the Parthenon in that no specific story is told. 15 Little interaction between figures exists and few narrative techniques are used. This is specific to the temple, and not ionic buildings as a whole. 10. Http://www. Ancient-Greece. Org/architecture/Athena-Nikkei. HTML 1 1 . Robin Francis Rhodes, Architecture and Meaning on the Athenian Acropolis, Cambridge University Press, First Edition, 1998. P. 113 12. Henry Sterile, Greece; From Mycenae to the Parthenon, Teaches Press, First Edition, 2001. P. 204 13. A. W. Lawrence, Greek Architecture, Yale University Press, Fifth Edition, 1996. P. 118 14. Joseph Rockery, The Dancing Column; On Order in Architecture, The MIT Press, First Edition, 1999. P. 230 15. Robin Francis Rhodes, Architecture and Meaning on the Athenian Acropolis, Cambridge University Press, First Edition, 1998. P. 120 ? ¬@lllustration 6: Athens, Temple of Athena Nikkei. ? ¬? ¬@Although originally based on Greek architecture, Roman temples developed so hat the Romans maintained their own distinct style. Roman temples’ orientations were, and still are, dominated by an axis, and the position of the temple in relation to the forum and the way that it is approached is an important stylistic feature. Roman temples were placed at the end of a clearly defined space. Set high on a podium, it could only be approached from the front. Greek temples differed, however, in that they were set down in an open area and approached from all sides. 16 The Romans favored the Corinthian order when building columns, as the ornamentation was an important factor in Roman architecture. As their style developed, so did the Roman arch, a very prominent aspect of many Roman buildings, including temples. 16. Leland M. Roth, Understanding Architecture; Its Elements, History, and Meaning, Western Press, Second Edition, 2007. P. 50 ? ¬The Manson Carr ©e, found in Ames in southern France, is constructed according to the Corinthian Order 17, as is seen by the presence of the Corinthian pillars, and is among the most well-preserved Roman temples of the Roman Empire. 18 The English translation is â€Å"square-house†, and is so-called because of its rectangular shape. 19 Built by Grippe in 16 BCC, the civic Roman temple was dedicated to his two sons who both died y oung, in an effort to show the loyalty and allegiance of the Roman colony to the imperial dynasty at the time. 0 The temple was the dominant structure in the forum of the Roman city at the time, serving as a reminder of the values that it represented to the city’s inhabitants. Although including elements consistent with an Etruscan style, very prominent Greek elements were present, showing the strong influence of Greek architecture, as opposed to Roman architecture which tended towards rounded arches and domes. 1 Twenty engaged columns can be found embedded along the walls of the calla, and delicate ornamental relief carvings of acanthus leaves and rosettes, found Just beneath a row of considerably fine dentists, adorn the frieze. 2 The reason that the temple is so well-preserved today is owed to the fact that it was rededicated as a Christian church during the fourth century, saving it from the destruction inflicted on other temples after Christianity was adopted as the offi cial state religion of Rome,23 leading us to believe that religion was a particularly prominent aspect of Roman ultra and, by extension, of the lives of the Romans themselves. ? ¬17. Http://www. Britannica. Com/Upchucked/topic/358799/Manson-Career 18. Http:// www. Sacred-destinations. Com/France/mimes-Manson-career 19. Http:// www. Formers. Mom/destinations/mimes/A30228. HTML 20. Http://www. Groundbreaking. Com/buildings/Manson_Career. HTML 21 . Http:// www. Reconstruction’s. Org/Manson-career-mimes 22. Http://en. Wisped. Org/wick/Manson_CarrÃÆ'CA#Awehitecture 23. httpHttpshelshelledm/ComaIsMansonrcarryeCAaAweeon in Rome, Italy, is a sthrong exstrongf an ancient Roman temple. It is worth noting that it was built as a temple to all gods, around 118 CE. 24 Semicircular arches, each of which compose two rings of brick spanning about 5 m, are found on the building’s exterior between the ends of both horizontal ranges of great conical vaults. The architect most likely thought of niches before piers, based on the Roman habit of expanding centralisecentralizedth both diagonal and cardinal recesses. 25 The largest and, arguably, most impressive element of the Pantheon is its dome. Domes were often used by the ancient Romans as space was maximised maximizedme acted moma symbol for the overarching heavens. 26 The clear span of the dome is 43. 3 m. 27 The Romans were aware of the large weights of the materials they were using and because of this they gradually decreased the thickness of the dome as it increased in height. How to cite The temple architecture of ancient Greece and Rome, Papers

History of Advertising free essay sample

In the 16th century some companies had a trade mark which was a two or three dimensional picture or sign. In both volume and technique, advertising has made its greatest advances in the u S. In the early stages Of U. S. Advertising it was hard and expensive to advertise nationally because the U. S. Was still undeveloped and there was little of no means of transport, distribution or communication. Eventually certain type of manufactures thought of the idea of bypassing wholesalers, retailers and using catalogs.Mail orders and amplest appeared around the sasss. Late in the 19th century many American firms began to market packaged goods under brand names. Previously consumers had not been aware of or influenced by brand names. The first product that had brand names were soap products. In the 1 asss a few brands came out and they were Ivory, Pears, Spoil, Colgate, Kirks American Family and Packers. We will write a custom essay sample on History of Advertising or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Not long after brands such as Royal baking powder, Quaker oats, Bakers chocolate, Hires root beer, Regal shoes and Watermans pens were nationally advertised.In the early sasss America began to become aware of such brand names like Bon Aim, Wrigley and Coca- Cola. After World War 1 advertising developed into a business so big that it was almost a trademark of America itself through the eyes of the rest of the world. This was expanded by technical improvements which made transportation, communication and graphics work easier, cheaper and better. The invention of electricity led to the illuminated outdoor poster, photoengraving and other modern printing inventions helped both editorial ND advertising departments of printed journals.In the sasss the radio was invented and this developed a whole new technique of selling by voice. During World War 2 the American advertising industry founded the war advertising council this used modern advertising to strengthen the American war effort. The organization still continued after the war was over as Cite Advertising Council this was used to function for the publics interest. Printed and broadcasted media contributed millions of dollars worth of advertising time and space to such projects each year.The most significant Advertising development after the war was Television. Television forced the advertising industry to use better techniques of selling because they had to use visual and voice. Since Vics have been invented Advertisers have been under threat because some viewers edit out the commercials when recording the program.

Substance Abuse In The Workplace Essays - Drug Control Law

Substance Abuse in the Workplace As widespread drug use is on the rise, many employers have begun to worry about the performance of their employees. Absenteeism, injuries, loss of productivity, employee morale, theft and fatalities are just some of the causes of drug use in the workplace. The idea of drug testing among workers has developed from society's concern over a perceived increase in the use of drugs and the relation between drug use and impairment, with resultant risks to the worker, fellow workers and the public. As early as 1987, 21% of employers had instituted drug-testing programs. Employers have begun to think that mass drug tests are the answer to their problems. What many of these employers don't know is that there are many problems that surround drug testing at work. One of the biggest of these problems is whether or not it is constitutional to conduct drug tests on the employees. Employers fail to educate themselves with established or recent laws about drug testing in the workplace and about huma n rights. Also, mass, low-cost screening tests may not be reliable or valid. Alcohol testing does not differentiate casual drinking from alcohol dependence or alcoholism. Drug tests can create an untrustworthy environment for the employees. There are better ways to address substance abuse. Drug testing in the workplace is an important issue for all of Canada's labour force, regardless if it's you're first job or if you've had a steady job for 30 years. Many employees, who have had to subjugate themselves to degrading and demeaning drug tests, feel that these tests violate their constitutional rights. It is an infringement on their privacy. In order for the tests to make sure there is no specimen tampering there must be an administrator present to oversee every action the employee makes during their drug test. For tests such as hair and breath testing this does present a major problem, but for urine tests men and women alike are disturbed by the direct observation of their urine collection. Unfortunately, the Canadian Charter of Rights and Freedoms applies only to the laws and actions of the federal and provincial governments and their agencies. It does not apply to the policies and actions of private employers. The Charter therefore does not protect private sector employees from unreasonable drug testing. It is necessary to state that currently an employer can terminate an employee's job if the employee has been using illegal drug s and alcohol, but only if such use is not considered a disability. Alcohol or drug addiction can be viewed as a physical and/or mental disability. In Ontario, the Ontario Human Rights, Citizenship, and Multiculturalism Act prohibit employment discrimination based on disability. Employers have a responsibility to accommodate employees who are disabled. Drug testing has not been proven to be against the Canadian Human Rights Commission. "In order to institute a drug testing policy into a company which complies with human rights legislation, an employer must be able to demonstrate that the testing is related to job performance, and not just substance abuse." Many employees feel that drug testing is a way of discriminating against people who might have a drug and/or alcohol disability. An example of such discrimination is found in Entrop v. Imperial Oil Ltd. The Ontario Board of Inquiry found that Imperial Oil Limited discriminated against Martin Entrop, a senior operator at the Sarni a Refinery, because of a disability. The Board of Inquiry found that "under a new Alcohol and Drug Policy introduce in 1992, Imperial Oil employees in "safety-sensitive" positions were required to notify management if they currently had or had previously had a substance abuse problem." After Mr.Entrop heard that this policy was coming into effect he informed his employer that he had had an alcohol problem about ten years earlier, that he had attended Alcoholics Anonymous, and that he had abstained from using alcohol since 1984. Mr. Entrop had been an employee for seventeen years and he had had no problems at work that were related to substance abuse, but Imperial Oil's policy required that Mr.Entrop be immediately removed form his current position. This example clearly shows that it is discriminatory to terminate a person's job because of a past or

Prehistoric Life During the Silurian Period

Prehistoric Life During the Silurian Period The Silurian period only lasted 30 or so million years, but this period of geologic history witnessed at least three major innovations in prehistoric life: the appearance of the first land plants, the subsequent colonization of dry land by the first terrestrial invertebrates, and the evolution of jawed fish, a huge evolutionary adaptation over previous marine vertebrates. The Silurian was the third period of the Paleozoic Era (542-250 million years ago), preceded the Cambrian and Ordovician periods and succeeded by the Devonian, Carboniferous and Permian periods. Climate and Geography Experts disagree about the climate of the Silurian period; global sea and air temperatures may have exceeded 110 or 120 degrees Fahrenheit, or they might have been more moderate (only 80 or 90 degrees). During the first half of the Silurian, much of the earths continents were covered by glaciers (a holdover from the end of the preceding Ordovician period), with climatic conditions moderating by the start of the ensuing Devonian. The giant supercontinent of Gondwana (which was destined to break apart hundreds of millions of years later into Antarctica, Australia, Africa and South America) gradually drifted into the far southern hemisphere, while the smaller continent of Laurentia (the future North America) straddled the equator. Marine Life During the Silurian Period Invertebrates. The Silurian period followed the first major global extinction on earth, at the end of the Ordovician, during which 75 percent of sea-dwelling genera went extinct. Within a few million years, though, most forms of life had pretty much recovered, especially arthropods, cephalopods, and the tiny organisms known as graptolites. One major development was the spread of reef ecosystems, which thrived on the borders of the earths evolving continents and hosted a wide diversity of corals, crinoids, and other tiny, community-dwelling animals. Giant sea scorpions - such as the three-foot-long Eurypterus - were also prominent during the Silurian, and were by far the biggest arthropods of their day. Vertebrates. The big news for vertebrate animals during the Silurian period was the evolution of jawed fish like Birkenia and Andreolepis, which represented a major improvement over their predecessors of the Ordovician period (such as Astraspis and Arandaspis). The evolution of jaws, and their accompanying teeth, allowed the prehistoric fish of the Silurian period to pursue a wider variety of prey, as well as to defend themselves against predators, and was a major engine of subsequent vertebrate evolution as the prey of these fish evolved various defenses (like greater speed). The Silurian also marked the appearance of the first identified lobe-finned fish, Psarepolis, which was ancestral to the pioneering tetrapods of the ensuing Devonian period. Plant Life During the Silurian Period The Silurian is the first period for which we have conclusive evidence of terrestrial plants - tiny, fossilized spores from obscure genera like Cooksonia and Baragwanathia. These early plants were no more than a few inches high, and thus possessed only rudimentary internal water-transport mechanisms, a technique that took tens of millions of years of subsequent evolutionary history to develop. Some botanists speculate that these Silurian plants actually evolved from freshwater algae (which would have collected on the surfaces of small puddles and lakes) rather than ocean-dwelling predecessors. Terrestrial Life During the Silurian Period As a general rule, wherever you find terrestrial plants, youll also find some kinds of animals. Paleontologists have found direct fossil evidence of the first land-dwelling millipedes and scorpions of the Silurian period, and other, comparably primitive terrestrial arthropods were almost certainly present as well. However, large land-dwelling animals were a development for the future, as vertebrates gradually learned how to colonize dry land. Next: the Devonian Period

MAZMAT responses to Chemical, Biological and Nuclear Agents and Essay

MAZMAT responses to Chemical, Biological and Nuclear Agents and Incidents - Essay Example According to a compilation of information by Gurr and Cole (2005), there have been various incidents (threatened and actual) of nuclear, biological, and chemical incidents (NBC) that have been reported and documented in the years since NBC weapons have been invented. They cite that in 1999, the Jane’s Intelligence Bureau was able to establish that elements having loyalties to Osama bin Laden were able to acquire biological weapons through mail. There was no clear indication of what kind of weapon was actually acquired, but sources speculated that these biological elements included a supply of diseases such as ebola, anthrax, and salmonella. Interested terrorist groups were able to avail of some of these weapons which included a freight of botulinium toxin. Gurr and Cole (2005) also discuss that in 1998, about 200 people from the Joan Finney Office building in Wichita, Kansas, USA were evacuated when a state employee discovered a package containing white powder and the note enc losed claimed that the substance was anthrax. The substance was sent by the Brothers for the Freedom of Americans, a neo-Nazi militia group. The package was later found to be non-toxic (Gurr and Cole, 2005). In 1995, Larry Harris was able to acquire freeze dried bubonic plague bacteria from the American Type Culture Collection (ATCC) in Maryland. His plan to acquire the bacteria was foiled when he telephoned the ATCC to check where the package was. The ATCC became suspicious and called the Centers for Disease Control, who later recovered three vials of the bacteria in his glove compartment (Gurr and Cole, 2005). In 1992, Henry Pierce sprayed 10 people with what he claimed was anthraz. On investigation, the police later found the material not to be anthrax. In 1981, a group of environmentalists left contaminated soil in a bucket. The soil later tested positive for anthrax. No one was harmed by the incident as the

Research paper of fish Example | Topics and Well Written Essays - 750 words

Of fish - Research Paper Example Pacific cod is more abundant than Atlantic cod thus should be preferred as an alternative. When available, cod caught with long lines, as a preferable technique, should offer a better choice which in turn minimizes harm to the ecosystem. Certified organic farmed cod can also be used as a suitable option. A total of $150 million in relief has been set aside for the New England fishermen and two other fishing areas included in an early vision of the Hurricane Sandy relief bill that passed to reduce on the cod fishing. (Kurlansky p,356)This was done to offer alternatives to farmers who plunged into the sea and made 100 trips so that they reduce the trips made in the sea so as to allow regeneration of the cod fish stocks. Introduction and proliferation of equipment and technology led to the increase of landed fish. These new technologies affected the cod fish populations as they increased the area and depth they were fished the catching of uneconomical species of fish thus depleting the stocks of important predator and prey species. The cod fishery had thrived for hundreds of years before overfishing set in as a result of poor management systems that depleted the stocks so greatly that led to the collapse of the industry. In 1992, more than 35,000 people lost their jobs due to the disappearance of the cod fish thus leaving them at a state of despair. ( Cod Jigging Report in 2006-2011) Due to the great cod fish populations, the fishery was sustainable as the cod fish was seasonal thus there could be regeneration of the stocks. The fishing trends and demands in the market played a major role in its collapse due to the overfishing that ensued and poor management thus leading to its collapse. The farmed versions content of the feed has changed as well. Forage fish provides two essential products: fish meal, for protein, and fish oil, for omega-3 fatty acids which is a suitable alternative to the fish. The different environmental conditions, food requirements for the wi ld fish and the nature of the environment hinder the domestication of the wild fish. (Rose p,146)Wild fish require specific temperatures and conditions that would favor their growth and reproduction and also specific food to sustain their development and this cannot be provided in a domestic environment limiting their production in a domestic setting. The presence or absence of the cod in the ecosystem impacts the pelagic fish, the herring to the zooplankton and phytoplankton through a â€Å"trophic cascade.† The presence of cod can therefore decrease the intensity of the local alga blooms. What other species are affected by its decline? There is little hope as there is slow recovery of the cod stocks due to inadequate food supplies, cooling of the North Atlantic and poor genetic stock due to the overfishing of larger cod. Recent studies reveal that recovery of cod stocks are showing promises of resurgence, despite earlier thoughts of complete collapse. Severity of the collap se of the cod fishery can only be equated to the large populations that lost their livelihoods: estimated 35,000 fishers, were left unemployed. Though there was a smooth intervention by the government to save the situation through a program known as Northern Cod Adjustment and Recovery Program and later through the Atlantic Ground fish strategy. (DISCHNER) There is increased economic diversification, emphasis on education and emergence of a thriving

World Trade in Merchandise Essay Example | Topics and Well Written Essays - 1750 words

World Trade in Merchandise - Essay Example Between the months of June and September, the price index lost 11.5 percent in terms of the dollar. (United Nations, 2009, p.45). The reason that can be identified for contraction in trade is a financial crisis. There was slow growth in output by almost 2 percent and the probability to fall further was high as well. The impact of the decline in total world production was magnified in trade. Since the month of September 2008, the imports and exports of the major developed and the developing countries declined. (World Trade Organization, 2009). The share of developing countries in the total global trade started to rise. It was assumed that a ‘decoupling effect’ would emerge into the picture and the developing countries will be less exposed to the economic downturn. As all regions of the World are slowing at once, the decline in demand conditions is more widespread than that of the past. The second reason that can be accounted for is the presence of increasing global supply chains in total trade. In the production process, the goods are supposed to cross many boundaries and the components in the final product are considered each and every time they cross a boundary. The only way to avoid such kind of effect is to measure the transactions in trade on the basis of the added value at each stage of the process of production. Shortage of trade finance can be regarded as the third reason for the situation. Shortage of trade has lead to a shortage of trade. This problem is receiving attention from government and other international institutions. The role of the WTO has been like an honest broker. It brings the top players to work together that will ensure the availability and also the affordability of trade finance. Protection is yet another factor that contributed to a contraction in trade. Rises in the level of protection are threatening to the prospects of recovery and delay the downturn. In the long run, the aggravated protectionism policy is a source of concern. 2. â€Å"Free trade, one of the greatest blessings which a government can confer on a people, is in almost every country unpopular† Lord Macaulay (1800-1859). It is true then and arguably very true today. (a) Critically discuss THREE forms of non-tariff barriers used by governments to limit the free flow of trade and assess their possible effects on consumers in any market of your choice. Nontariff barriers The three forms of nontariff barriers are imported quota, import licensing requirements, and import deposits. American firms have registered few grievances against Dutch firms. The Dutch came up as the neutral traders of Europe as they opted for a level playing field for trade matters. Soft drinks, beer, and petroleum products are the items where excise tax is levied. The excise tax is borne by the importers in addition to customs duty. The European Union aims to create a single international market and harmonize the excise taxes. Nontariff barriers are a measure used by the government to favor goods that are produced domestically over goods produced in foreign countries. The nontariff barriers are used to reduce the volume of imports so as to help the domestic producers. A quota may be defined as the maximum limitation either in physical units or in other terms imposed on imports of a product for a certain period of time.

Physico-chemical Processes that Occur During Freezing

Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio Physico-chemical Processes that Occur During Freezing Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio